Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries

Evgeniya S Omelina,Anna E Letiagina,Alexey V Pindyurin,Anton V Ivankin

doi:10.1186/s12864-019-5847-2

Abstract

BackgroundMassively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study; ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. The sequences of BC–ROI combinations present in the libraries may be either known a priori or not. In the latter case, it is necessary to identify these combinations before performing functional experiments. Typically, this is done by PCR amplification of the BC–ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. However, chimeric DNA molecules formed on templates with identical spacer fragment during the amplification process may substantially hamper the identification of genuine BC–ROI combinations, and as a result lower the performance of the assays.ResultsTo identify settings that minimize formation of chimeric products we tested a number of PCR amplification parameters, such as conventional and emulsion types of PCR, one- or two-round amplification strategies, amount of DNA template, number of PCR cycles, and the duration of the extension step. Using specific MPRA libraries as templates, we found that the two-round amplification of the BC–ROI regions with a very low initial template amount, an elongated extension step, and a specific number of PCR cycles result in as low as 0.30 and 0.32% of chimeric products for emulsion and conventional PCR approaches, respectively.ConclusionsWe have identified PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries. In addition, we found that there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings.

Highlights

Parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants
It has been previously shown that routine conventional PCR coamplification of DNA sequences containing two variable motifs separated by a constant region frequently leads to formation of undesired chimeric molecules, from 5.4 to 30% [14,15,16,17,18,19,20,21,22]
Our findings indicate that synthesis of chimeric DNA molecules during amplification of BC–ROI fragments from a mixture of two different templates can be suppressed to almost negligible levels using two-round emulsion PCR (ePCR) with optimized parameters

Summary

Introduction

Parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. It has been previously shown that routine conventional PCR coamplification of DNA sequences containing two variable motifs (in the case of MPRAs, BC and ROI) separated by a constant region frequently leads to formation of undesired chimeric molecules, from 5.4 to 30% [14,15,16,17,18,19,20,21,22]. Such PCR products complicate and can mislead the MPRA data analysis as well as decrease the productivity of the approach, as the association of the same BC with different ROIs leads to the elimination of all such BCs and ROIs from the analysis. The emulsion PCR (ePCR) method, which provides a simple physical separation of the template DNA molecules due to the usage of water-in-oil emulsion [28], seems to be a solution of the problem

Methods

Results

Discussion

Conclusion